This invention relates to biologically active peptides. More particularly, this invention relates to biologically active peptides with reduced toxicity. Further, this invention relates to a method of modifying biologically active peptides, that are either N-terminally (amino-terminal) substituted or unsubstituted, to reduce their toxicity in animals.
In accordance with an aspect of the present invention, there is provided an unsubstituted biologically active peptide or protein. In another aspect, there is provided a N-terminal substituted peptide or protein having the formula: 
wherein X is a biologically active peptide or protein, and N is a nitrogen atom. The peptide or protein is preferably an ion channel-forming peptide or protein. T is a lipophilic moiety, and W is another T group or hydrogen.
The term xe2x80x9clipophilic,xe2x80x9d as used herein, means that the lipophilic moiety enhances the interaction of the peptide or protein with a lipid membrane, such as, for example, a cell membrane.
Lipophilic moieties which may be employed, include, but are not limited to, any moiety which may be placed on the N-terminal of the peptide through a condensation reaction with nitrogen. The lipophilic moiety T may be, for example, a carboxylic acid, a phosphoric acid, preferably an alkylphosphoric acid, a phosphonic acid, preferably an alkylphosphonic acid, a sulfonic acid, preferably an alkylsulfonic acid, or an alkyl group. Preferably T is: 
wherein R is a hydrocarbon having at least two and no more than 16 carbon atoms.
In one embodiment, R is an alkyl group. The alkyl group may be a straight chain or branched chain alkyl group, or a cycloalkyl group. For example, R may be CH3(CH2)nxe2x80x94, wherein n is from 1 to 14. Preferably, n is from 3 to 12, more preferably from 4 to 11, still more preferably from 6 to 11, and most preferably n is 6, whereby T is an octanoyl group.
In another embodiment, R is an aromatic (including phenyl and naphthyl), or an alkyl aromatic group. For example, R may be 
wherein z is from 0 to 6. Preferably, z is 1 or 2.
In another embodiment, R is 
wherein n is from 1 to 5. Preferably n is 1, whereby R is an ibuprofyl group.
In yet another embodiment, T is: 
wherein x is from 1 to 14. Preferably, x is 2, and T is a succinyl group.
In another embodiment, T is: 
wherein y is from 1 to 14. Preferably, y is 12, whereby T is a sphingosine group.
In yet another embodiment T is 
wherein x and y are hereinabove described. Preferably, x is 2, and y is 12.
In one embodiment, W is hydrogen.
Applicant has found that when biologically active peptides have substitutions at the N-terminal such as those hereinabove described, such peptides have increased biological activity against target cells, viruses, and virally-infected cells, as compared with unsubstituted peptides or peptides substituted at the N-terminal with an acetyl group. Applicant also has found that the N-terminal substitutions hereinabove described significantly increase the biological activity of xe2x80x9cshortxe2x80x9d peptides, i.e., peptides having no more than 14 amino acid residues.
In another embodiment of this invention the biologically active peptides are not N-terminally substituted and have broad spectrum anti-tumor and anti-microbial activity.
As hereinabove stated, the biologically active peptides or proteins of the present invention are preferably ion channel-forming peptides. An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the permeability for ions across a natural or synthetic lipid membrane. B. Christensen, et al., PNAS Vol. 85, pgs. 5072-5076 (July 1988) describes a methodology which indicates whether or not a peptide or protein has ion channel-forming properties and is therefore an ionophore. As used herein, an ion channel-forming peptide or ion channel-forming protein is a peptide or protein which has ion channel-forming properties as determined by the method of Christensen, et al. This Christensen article is entirely incorporated herein by reference.
An amphiphilic peptide or protein is a peptide or protein which includes both hydrophobic and hydrophilic peptide or protein regions.
The ion channel-forming peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water. In addition, the structure of such peptides provides for flexibility of the peptide molecule. Such peptides are capable of forming an alphahelical structure. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rodlike structure.
In general, such peptides have at least 7 amino acids, and in many cases have at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids.
The peptides and/or analogues or derivatives thereof may be administered to a host, for example a human or non-human animal, in an amount effective to inhibit growth of a target cell, virus, or virally-infected cell. Thus, for example, the peptides and/or analogues or derivatives thereof may be used as anti-microbial agents, anti-viral agents, anti-bacterial agents, anti-tumor agents, anti-parasitic agents, and spermicides, as well as agents exhibiting other bioactive functions.
The term xe2x80x9canti-microbialxe2x80x9d as used herein means that the peptides or proteins of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes, such as bacteria, fungi, viruses, or the like.
The term xe2x80x9canti-bacterialxe2x80x9d as used herein means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological functions of bacteria, including death, destruction, or prevention of the growth or proliferation of the bacteria when contacted with the peptides or proteins.
The term xe2x80x9cantibioticxe2x80x9d as used herein means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological functions of the non-host cell, tissue, or organism, including death, destruction, or prevention of the growth or proliferation of the non-host cell, tissue, or organism when contacted with the peptides or proteins.
The term xe2x80x9cspermicidalxe2x80x9d as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the motility of sperm.
The term xe2x80x9canti-fungalxe2x80x9d as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of fungi.
The term xe2x80x9canti-viralxe2x80x9d as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of viruses, or of virally-infected cells.
The term xe2x80x9canti-tumorxe2x80x9d as used herein means that the peptides or proteins inhibit the growth of or destroy tumors, including cancerous tumors.
The term xe2x80x9canti-parasiticxe2x80x9d as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of parasites.
The peptides or proteins of the present invention have a broad range of potent anti-tumor and antibiotic activity against a plurality of tumor types and microorganisms, including gram-positive and gram-negative bacteria, fungi, protozoa, and the like, as well as parasites. The peptides or proteins of the present invention allow a method for treating or controlling tumor growth and microbial infection caused by organisms which are sensitive to the peptides or proteins. Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an anti-tumor or anti-microbial amount of at least one of the peptides or proteins.
In another embodiment of this invention, methods are provided for reducing the toxicity of unmodified peptides or of N-terminally modified peptides in a host without reducing the anti-tumor or anti-microbial activity of the peptides. This method includes forming a methane sulfonate derivative or analogue of an unsubstituted peptides or N-terminal substituted peptide having the formula: 
wherein X is a biologically active peptide or protein, the peptide being an ion channel-forming peptide or protein, T is a lipophilic moiety or hydrogen. The anti-tumor or anti-microbial activity of the methane sulfonate derivative or analogue of the unsubstituted or N-terminal substituted peptide is not reduced or is not significantly reduced as compared to the corresponding peptide not derivatized with a methane sulfonate group. The phrase xe2x80x9cnot significantly reduced,xe2x80x9d as used in this application, means that the methane sulfonate derivatives or analogues retain anti-tumor or anti-microbial activity of the underivatized compounds. Preferably, the methane sulfonate derivatives or analogues retain at least 50%, and preferably 75% or more, of the anti-tumor or anti-microbial activity of the underivatized compounds.
In one embodiment of the invention, the methane sulfonate derivative can be formed by suspending a free base of the unsubstituted or N-terminal substituted peptide in water, and then mixing the suspended peptide and at least 0.5 equivalents of a sodium bisulfite-formaldehyde complex (or other suitable bisulfite-formaldehyde complex) for each free amino group in the peptide. This reaction proceeds for a period of 10 minutes or more to produce the methane sulfonate derivative or analogue of the peptide.
The amount of the bisulfite-formaldehyde complex can be varied without departing from the invention. Preferably, the suspended free base is mixed with 0.5 to 10 equivalents of the bisulfite-formaldehyde complex for each free amino group in the peptide. The use of 1 to 5 equivalents is preferred, and 1.1 to 3 equivalents is particularly preferred.
In the process according to this embodiment of the invention, the mixing period also can be varied widely.
Preferably the peptide free base and complex are mixed for a period of 10 minutes to 2 hours, or 10 minutes to 1 hour. A mixing period in the range of from 15 minutes to 30 minutes is particularly preferred.
The starting free base of the unsubstituted peptide or N-terminal substituted peptide can be prepared by neutralizing a salt of the peptide. This neutralization can take place by treating the salt with a base solution, such as a sodium carbonate solution. After neutralization, the free base peptide, which may precipitate, can be isolated prior to suspending it in the water.
After the reaction procedure is completed, the methane sulfonate derivative or analogue of the peptide product can be recovered, e.g., by filtering. Additionally, this product can be lyophilized, if desired.
One method according to this embodiment of the invention can be summarized by the reaction equation provided below: 
wherein X is a biologically active peptide or protein, N is a nitrogen atom and T is a lipophilic moiety or hydrogen. The peptide or protein is preferably an ion channel-forming peptide or protein.
In another embodiment or the invention the methane sulfonate derivative is formed by mixing the peptide salt with 12.5 equivalents of 30% formaldehyde solution and 6.25 equivalents of 1M sodium bicarbonate solution for each amino group in the peptide. The adduct of the peptide with formaldehyde precipitates and can be collected by centrifugation of filtration. The sodium methane sulfonate derivative is then formed by mixing the formaldehyde adduct with sodium metabisulfite.
The methane sulfonate analogue of the peptide is shown to have reduced toxicity in vivo, but retains its anti-tumor and anti-microbial activity.
Because of the antibiotic, anti-microbial, anti-viral, and anti-bacterial properties of the peptides or proteins, they may also be used as preservatives, sterilants, or disinfectants of materials susceptible to microbial or viral contamination.
The peptide or proteins and/or derivatives or analogues thereof may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, a non-toxic buffer, or a physiological saline solution. Such pharmaceutical compositions may be used topically or systemically and may be in any suitable form such as a liquid, solid, semi-solid, injectable solution, tablet, ointment, lotion, paste, capsule, or the like. The peptide or protein compositions may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa, viruses, and the like, as well as by parasites.
The peptides or proteins of the present invention may be administered to a host, in particular a human or non-human animal, in an effective antibiotic and/or anti-tumor and/or anti-fungal and/or anti-viral and/or anti-microbial and/or anti-bacterial and/or anti-parasitic and/or spermicidal amount.
Depending on the use, a composition in accordance with the invention will contain an effective anti-microbial amount and/or an effective spermicidal amount and/or an effective anti-fungal amount and/or an effective anti-viral amount and/or an effective anti-tumor amount and/or an effective anti-parasitic amount and/or an effective antibiotic amount of one or more of the peptides or proteins of the present invention which have such activity. The peptides or proteins may be administered by direct application of the peptides or proteins to the target cell, virus, or virally-infected cell, or indirectly applied through systemic administration.
The peptides or proteins of the present invention may also be employed in promoting or stimulating healing of a wound in a host.
The term xe2x80x9cwound healingxe2x80x9d as used herein includes various aspects of the wound healing process. These aspects include, but are not limited to, increased contraction of the wound, increased deposition of connective tissue, as evidenced by, for example, increased deposition of collagen in the wound, and increased tensile strength of the wound, i.e., the peptides or proteins increase wound breaking strength. The peptides or proteins of the present invention may also be employed so as to reverse the inhibition of wound healing caused by conditions which depress or compromise the immune system.
The peptides or proteins of the present invention may be used in the treatment of external burns and to treat and/or prevent skin and burn infections. In particular, the peptides or proteins may be used to treat skin and burn infections caused by organisms such as, but not limited to, P. aeruginosa and S. aureus. 
The peptides or proteins are also useful in the prevention or treatment of eye infections. Such infections may be caused by bacteria such as, but not limited to, P. aeruginosa, S. aureus, and N. gonorrhoeae, by fungi such as, but not limited to, C. albicans and A. fumigatus, by parasites such as, but not limited to, A. castellani, or by viruses.
The peptides or proteins may also be effective in killing cysts, spores, or trophozoites of infection-causing organisms. Such organisms include, but are not limited to Acanthamoeba, which form trophozoites or cysts, C. albicans, which form spores, and A. fumigatus, which also form spores.
The peptides or proteins of the present invention may also be employed in the treatment of tumors. In general, they are active against tumors that arise in tissues and organs such as, but not limited to, breast, lung, colon, rectum, cervix, ovaries prostate, stomach, as well as melanoma and leukemias. More specifically, they are active against non-small cell lung carcinomas and adenocarcinomas of, for example, the breast, cervix, prostate, lung, colon, rectum, stomach, and ovaries. In addition, the peptides and proteins of the present invention are active against tumors that are resistant to other anti-tumor agents. A significant reason for resistance, the removal of anti-tumor agents by the efflux pump, is not believed to apply to the peptides and proteins of the present invention which are believed to function via the unique mechanism of forming ion channels.
The peptides or proteins may also be administered to plants in an effective anti-microbial or anti-viral or anti-parasitic amount to prevent or treat microbial, viral, or parasitic contamination thereof.
The peptides or proteins of the present invention may also be employed in the treatment of serious lung infections. In particular, the peptides or proteins may be used to treat lung infections caused by organisms such as, but not limited to, P.aeruginosa in cystic fibrosis patients. In general, in the treatment of such infections, the peptides or proteins are administered to a subject in need of treatment by an inhalation procedure. For example, the active peptide or protein ingredient can be delivered by inhalation using either a nebulizer, metered dose inhaler (MDI), or a dry powder inhaler (DPI).
In employing such compositions by inhalation, the active peptide or protein is present in the following amounts: for a nebulizer, a solution of between 5 and 200 mg/ml; for MDI or DPI, an amount between 0.5 and 45 mg.
The peptides or proteins also may be administered to a subject for treating sepsis, septic shock, and other related ailments, in that such peptides neutralize bacterial endotoxins. In general, the peptides or proteins are positively charged, while, in general, the bacterial endotoxins are negatively charged. The peptides or proteins are particularly useful in that such compounds neutralize bacterial endotoxins without neutralizing essential proteins in plasma (such as heparin, for example).
xe2x80x9cTreat,xe2x80x9d xe2x80x9ctreated,xe2x80x9d or xe2x80x9ctreating,xe2x80x9d as used in this application, may mean complete elimination of a disease, ailment, or symptoms, or it may mean reducing, suppressing, or ameliorating the severity of the disease, ailment, or symptoms.
The peptides or proteins, when used in topical compositions, are generally present in an amount of at least 0.1%, by weight. In most cases, it is not necessary to employ the peptide in an amount greater than 2.0%, by weight.
In employing such compositions systemically (intramuscular, intravenous, intraperitoneal), the active peptide or protein is present in an amount to achieve a serum level of the peptide of at least about 5 xcexcg/ml. In general, the serum level of peptide or protein need not exceed 500 xcexcg/ml. A preferred serum level is about 100 xcexcg/ml. Such serum levels may be achieved by incorporating the peptide or protein in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg. In general, the peptide(s) or protein(s) need not be administered at a dose exceeding 100 mg/kg.
The peptides or proteins may be produced by known techniques and obtained in substantially pure form. For example, the peptides may be synthesized on an automatic peptide synthesizer. Journal of the American Chemical Society, Vol. 85, pgs. 2149-54 (1963) (which article is entirely incorporated herein by reference). It also is possible to produce such peptides or proteins by genetic engineering techniques. The codons encoding specific amino acids are known to those skilled in the art, and therefore, DNA encoding the peptides may be constructed by appropriate techniques, and one may clone such DNA into an appropriate expression vehicle (e.g., a plasmid) which is transfected into an appropriate organism for expression of the peptide or protein.
In one embodiment of the invention, upon production or synthesis of the peptide or protein, the N-terminal (NH2 or amino terminal) of the peptide is reacted such that the lipophilic moiety is attached to the N-terminal of the peptide. For example, the reaction may be a condensation reaction with an amine. When the lipophilic moiety T is 
the N-terminal is reacted with a carboxylic acid of the formula Rxe2x80x94COOH, wherein R is a hydrocarbon having at least 2 carbon atoms. The reaction may be carried out in the presence of a coupling agent, such as, for example, DCC, or DIC, and HOBT, or in the presence of an acid chloride. Such a reaction results in the formation of an N-terminal substituted peptide or protein having the structural formula hereinabove described.
In one embodiment, X is a peptide which is a basic (positively charged) polypeptide having at least sixteen amino acids, wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids. Still more particularly, the hydrophobic amino acids are in groups of two adjacent amino acids, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino acids by at least one amino acid other than a hydrophobic amino acid (preferably at least two amino acids) and generally by no greater than four amino acids, and the amino acids between pairs of hydrophobic amino acids may or may not be hydrophilic.
The hydrophilic amino acids are generally also in groups of two adjacent amino acids in which at least one of the two amino acids is a basic hydrophilic amino acid, with such groups of two hydrophilic amino acids being spaced from each other by at least one amino acid other than a hydrophilic amino acid (preferably at least two amino acids) and generally no greater than four amino acids, and the amino acids between pairs of hydrophilic amino acids may or may not be hydrophobic.
In accordance with a particularly preferred embodiment, the polypeptide comprises a chain of at least four groups of amino acids, with each group consisting of four amino acids. Two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are hydrophilic, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other being a basic or neutral hydrophilic amino acid.
The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha). The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser, Thr, and homoserine (Hse). The basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginone (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C, or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different. In one embodiment, the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C, and D may be the same in some or all of the groups or may be different in some or all of the groups.
The polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids. It is to be understood, however, that the polypeptide does not have to consist entirely of the groups described above. The polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention.
The groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary, provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted.
Thus, the biologically active polypeptide may comprise a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
In one embodiment, each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, one of C or D is a basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid. The resulting polypeptide chain, therefore, may have one of the following sequences:
(X1)a(A-B-C-D)n(Y1)b 
(X2)a(B-C-D-A)n(Y2)b 
(X3)a(C-D-A-B)n(Y3)b 
(X4)a(D-A-B-C)n(Y4)b 
wherein X1 is D or C-D- or B-C-D-;
Y1 is -A or -A-B or -A-B-C;
X2 is A-, D-A- or C-D-A-;
Y2 is -B, -B-C or B-C-D;
X3 is B-, A-B-, D-A-B-;
Y3 is -C, -C-D, -C-D-A;
X4 is C-, B-C-, A-B-C-;
Y4 is -D, -D-A, -D-A-B;
a is 0 or 1; b is 0 or 1; and n is at least 4.
It is to be understood that the peptide chain may include amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and does not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted groups of four amino acids are not spaced from each other.
As representative examples of such peptides, the following may be mentioned:
I Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys (SEQ ID NO:1)
II Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys. (SEQ ID NO:2)
III Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala- (SEQ ID NO:3)
IV Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe- (SEQ ID NO:4)
V Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser (SEQ ID NO:5)
The peptide may have amino acids extending from either end of the chain. For example, the chains may have a Ser-Lys sequence before the xe2x80x9cAlaxe2x80x9d end, and/or an Ala-Phe sequence after the xe2x80x9cLysxe2x80x9d end. Other amino acid sequences may also be attached to the xe2x80x9cAlaxe2x80x9d and/or the xe2x80x9cLysxe2x80x9d end.
Similarly, in any polypeptide chain having at least four groups of amino acids of the sequence as described above, the chain may have, for example, a C-D sequence before the first A-B-C-D group. Also other amino acid sequences may be attached to the xe2x80x9cAxe2x80x9d and/or the xe2x80x9cDxe2x80x9d end of one of these polypeptide chains. Also, there may be amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other.
In accordance with another embodiment, X is a magainin peptide.
A magainin peptide is either a magainin such as magainin I, II, or III, or an analogue or derivative thereof. The magainin peptides preferably include the following basic peptide structure X12:
R11-R11-R12-R13-R11-R14-R12-R11-R14-R12-R11-R11-R11-R14a-(R15)n-R14a-R14 
wherein R11 is a hydrophobic amino acid; R12 is a basic hydrophilic amino acid; R13 is a hydrophobic, neutral hydrophilic, or basic hydrophilic amino acid; R14 and R14a are hydrophobic or basic hydrophilic amino acids; R15 is glutamic acid or aspartic acid; and n is 0 or 1. In a preferred embodiment, R13 is a hydrophobic or neutral hydrophilic amino acid, R14a is a hydrophobic amino acid, and R15 is glutamic acid or aspartic acid.
Thus, for example, a magainin peptide may include the following structure:
-Y12-X12-
where X12 is the hereinabove described basic peptide structure, and Y12 is
(i) R12 
(ii) R14a-R12 
(iii) R11-R14a-R12 
(iv) R14-R11-R14a-R12 
where R11, R12, R14, and R14a are as previously defined.
A magainin peptide may also have the following structure:
-X12-Z12-
wherein X12 is as previously defined and Z12 is:
(i) R16, where R16 is a basic hydrophilic amino acid or asparagine or glutamine; or
(ii) R16-R17, where R17 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid.
Preferably, R17 is a basic hydrophilic amino acid.
A magainin peptide may also have the following structure:
(Y12)a-X12- Z12)b 
where X12, Y12, and Z12 are as previously defined, a is 0 or 1, and b is 0 or 1.
As representative examples of such peptides, the following may be mentioned:
I Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys (SEQ ID NO: 154)(OH) or (NH2)
II Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Met-Lys-Lys (SEQ ID NO: 155)(OH) or (NH2)
The magainin peptides may also include the following basic peptide structure X13:
R14-R11-R14a-R12-R11-R11-R12-R13-R11-R14-R12-R11-R11-R12,
wherein R11,R12,R13,R14, and R14a are amino acids as hereinabove described.
The magainin peptide may also include the following structure: -X13-Z13-,
wherein X13 is the hereinabove described basic peptide structure and Z13 is
(R11)n-(R11)n-(R11)n-(R14a)n-(R15)n-(R14a)n-(R14)n-(R16)n-(R17)n,
wherein R11, R14, R14a, R15 R16, and R17 are as hereinabove described, and n is 0 or 1, and each n may be the same or different.
The magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids. A magainin peptide preferably has 22 or 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include additional amino acids at the amino end or at the carboxyl end, or at both ends.
As representative examples of such magainin peptides, there may be mentioned peptides having the following primary sequences as given in the accompanying sequence listing, as well as appropriate analogues and derivatives thereof:
The following are examples of peptide derivatives or analogs of the basic structure:
Magainin peptides are described in Proc. Natl. Acad Sci. Vol. 84 pp. 5449-53 (Aug. 87), which article is entirely incorporated herein by reference. The term xe2x80x9cmagainin peptidesxe2x80x9d as used herein refers to the basic magainin structure as well as derivatives and analogs thereof, including, but not limited to, the representative derivatives or analogs.
In accordance with a further embodiment, X may be a PGLa peptide or an XPF peptide. A PGLa peptide is either PGLa or an analogue or derivative thereof. The PGLa peptides preferably include the following basic peptide structure X14:
R11-R17-R12-R11-R14-R14-R14-R12-R11-R11-R12-R11-R11-R12,
where R11, R12, R14, and R17 are as previously defined.
The PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl ends.
Thus, for example, a PGLa peptide may have the following structure:
-Y14-X14-
where X14 is as previously defined and
Y14 is
(i) R11; or
(ii) R14-R11,
where R11 and R14 are as previously defined.
For example, a PGLa peptide may also have the following structure:
-X14-Z14-
where X14 is as previously defined; and Z14 is:
(i) R11; or
(ii) R11,-R11,
where R11 is as previously defined.
A PGLa peptide may also have the following structure:
(Y14)a-X14-(Z14)b 
where X14, Y14, and Z14 are as previously defined, a is 0 or 1, and b is 0 or 1.
An XPF peptide is either XPF or an analogue or derivative thereof. The XPF peptides preferably include the following basic peptide structure X16:
R11-R17-R12-R11-R14-R18-R17-R11-R14-R12-R11-R11-R12-R11-R11-R11-R11-R12-(R15)n-R11,
wherein R11, R12, R14, R15, and R17 are as previously defined; R18 is glutamine, asparagine, a basic hydrophilic amino acid, or a hydrophobic amino acid; and n is 0 or 1.
The XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids. Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino end, or at the carboxyl end, or at both the amino and carboxyl ends.
Thus, for example, an XPF peptide may include the following structure:
-Y16-X16-
where X16 is as previously defined and Y16 is
(i) R11, or
(ii) R14-R11,
where R11 and R14 are as previously defined.
An XPF peptide may include the following structure:
-X16-Z16-
where X16 is as previously defined and Z16 is:
(i) R11;
(ii) R11-R18;
(iii) R11-R18-Proline; or
(iv) R11-R18-Proline-R12, wherein R11, R12, and R18 are as previously defined.
An XPF peptide may also have the following structure:
(Y16)a-X16-(Z16)b 
where X16, Y16, and Z16 are as previously defined, a is 0 or 1, and b is 0 or 1.
Preferred are XPF or PGLa peptides, which are characterized by the following primary amino acid sequences, as given in the accompanying sequence listing:
PGLa: (SEQ ID NO:12) (NH2)
XPF: (SEQ ID NO:13)
A review of XPF and PGLa can be found in Hoffman et al, EMBO J., 2:711-714, 1983; Andreu, et al, J. Biochem., 149:531-535, 1985; Gibson, et al J. Biol. Chem., 261:5341-5349, 1986; and Giovannini, et al, Biochem. J., 243:113-120, 1987. These articles each are entirely incorporated herein by reference.
In accordance with yet another embodiment, X is a CPF peptide or an appropriate analogue or derivative thereof.
CPF peptides, as well as analogues and derivatives thereof, are herein sometimes referred to collectively as CPF peptides.
The CPF peptide may be one which includes the following basic peptide structure X20:
R21-R21-R22-R22-R21-R21-R23-R21-R21-R21-R23-R21-R21-R24-R25-R21,
wherein R21 is a hydrophobic amino acid;
R22 is a hydrophobic amino acid or a basic hydrophilic amino acid;
R23 is a basic hydrophilic amino acid;
R24 is a hydrophobic or neutral hydrophilic amino acid; and
R25 is a basic or neutral hydrophilic amino acid.
The hereinabove basic structure is hereinafter symbolically indicated as X20.
The hydrophobic amino acids are Ala, Cys, Phe, Gly, Ile, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
The neutral hydrophilic amino acids are Asn, Gln, Ser, Thr, and homoserine (Hse).
The basic hydrophilic amino acids are Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
The CPF peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino and/or carboxyl ends or both the amino and carboxyl ends. In general, the peptide does not include more than 40 amino acids.
The CPF peptides including the above basic structure preferably have from 1 to 4 additional amino acids at the amino end.
Accordingly, such preferred peptides may be represented by the structural formula:
Y20-X20-
wherein X20 is the hereinabove described basic peptide structure and Y20 is
(i) R25-;
(ii) R22-R25-;
(iii) R21-R22-R25;
(iv) R22-R21-R22-R25; or preferably
(v) Glycine -R21-R22-R25,
wherein R21, R22, and R25 are as previously defined.
The carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.
In a preferred embodiment, the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:
-X20-Z20, wherein
X20 is the hereinabove defined basic peptide structure and Z20 is
(ii) R21-R21-;
(iii) R21-R21-R24;
(iv) R21-R21-R24-R24;
(v) R21-R21-R24-R24-R26;
(vi) R21-R21-R24-R24-R26-Gln; or
(vii) R21-R21-R24-R24-R26-Gln-Gln, wherein R21 and R24 are as previously defined, and R26 is proline or a hydrophobic amino acid.
Preferred peptides may be represented by the following structural formula
(Y20)a-X20-(Z20)b 
wherein X20, Y20 and Z20 are as previously defined, a is 0 or 1, and b is 0 or 1.
Representative examples of CPF peptides which may be employed, some of which have been described in the literature, include the following sequences as given in the accompanying sequence listing:
(SEQ ID NO:14)
(SEQ ID NO:15)
(SEQ ID NO:16)
(SEQ ID NO:17)
(SEQ ID NO:18)
(SEQ ID NO:19)
(SEQ ID NO:20)
(SEQ ID NO:21)
(SEQ ID NO:22)
(SEQ ID NO:23)
(SEQ ID NO:24)
(SEQ ID NO:25)
(SEQ ID NO:26)
A review of the CPF peptides can be found in Richter, K., Egger, R., and Kreil (1986) J. Biol. Chem., 261, 3676-3680; Wakabayashi, T., Kato, H., and Tachibaba, S. (1985) Nucleic Acids Research, 13, 1817-1828; and Gibson, B. W., Poulter, L., Williams, D. H., and Maggio, J. E. (1986) J. Biol. Chem., 261, 5341-5349. These articles each are entirely incorporated herein by reference.
In accordance with yet another embodiment, X is a peptide which includes one of the following basic structures X31 through X37 wherein:
X31 is -[R31-R32-R32-R33-R31-R32-R32]-n;
X32 is -[R32-R32-R33-R31-R32-R32-R31]-n;
X33 is -[R32-R33-R31-R32-R32-R31-R32]-n;
X34 is -[R33-R31-R32-R32-R31-R32-R32]-n;
X35 is -[R31-R32-R32-R31-R32-R32-R33]-n;
X36 is -[R32-R32-R31-R32-R32-R33-R31]-n; and
X37 is -[R32-R31-R32-R32-R33-R31-R32]-n;
wherein R31 is a basic hydrophilic amino acid, R32 is a hydrophobic amino acid, R33 is a neutral hydrophilic, basic hydrophilic, or hydrophobic amino acid, and n is from 1 to 5.
The basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp and Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser, Thr, and homoserine (Hse).
In accordance with one embodiment, when the peptide includes the structure X31, the peptide may include the following structure:
Y31-X31, wherein X31 is as hereinabove described, and Y31 is:
(i) R32;
(ii) R32-R32;
(iii) R31-R32-R32;
(iv) R33-R31-R32-R32;
(v) R32-R33-R31-R32-R32; or
(vi) R32-R32-R33-R31-R32-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X31, the peptide may include the following structure:
X31-Z31, wherein X31 is as hereinabove described, and Z31 is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R33;
(v) R31-R32-R32-R33-R31; or
(vi) R31-R32-R32-R33-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y31)a-X31-(Z31)b, wherein Y31 and Z31 are as previously defined, a is 0 or 1, and b is 0 or 1.
When the peptide includes the structure X32, the peptide may include the following structure:
Y32-X32, wherein X32 is as hereinabove described, and Y32 is:
(i) R31;
(ii) R32-R31;
(iii) R32-R32-R31;
(iv) R31-R32-R32-R31;
(v) R33-R31-R32-R32-R32; or
(vi) R32-R33-R31-R32-R32-R31, wherein R31, R32, and R33 are as hereinabove described.
In another embodiment, when the peptide includes the structure X32, the peptide may include the following structure:
X32-Z32, wherein X32 is as hereinabove described, and Z32 is:
(i) R32;
(ii) R32-R32;
(iii) R32-R32-R33;
(iv) R32-R32-R33-R31;
(v) R32-R32-R33-R31-R32; or
(vi) R32-R32-R33-R31-R32-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y32)a-X32-(Z32)b, wherein Y32 and Z32 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with another embodiment, when the peptide includes the structure X33, the peptide may include the following structure:
Y33-X33, wherein X33 is as hereinabove described, and Y33 is:
(i) R32;
(ii) R31-R32;
(iii) R32-R31-R32;
(iv) R32-R32-R31-R32;
(v) R31-R32-R32-R31-R32; or
(vi) R33-R31-R32-R32-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X33, the peptide may include the following structure:
X33-Z331 wherein X33 is as hereinabove described, and Z33 is:
(i) R32;
(ii) R32-R33;
(iii) R32-R33-R31;
(iv) R32-R33-R31-R32;
(v) R32-R33-R31-R32-R32; or
(vi) R32-R33-R31-R32-R32-R31, wherein R31, R32, and R33 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y33)a-X33-(Z33)b, wherein X33, Y33, and Z33 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with yet another embodiment, when the peptide includes the structure X34, the peptide may include the following structure:
Y34-X34, wherein X34 is as hereinabove described, and Y34 is:
(i) R32;
(ii) R32-R32;
(iii) R31-R32-R32;
(iv) R32-R31-R32-R32;
(v) R32-R32-R31-R32-R32; or
(vi) R31-R32-R32-R31-R32-R32, wherein R31 and R32 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X34, the peptide may include the following structure:
X34-Z34, wherein X34 is as hereinabove described, and Z34 is:
(i) R33;
(ii) R33-R31;
(iii) R33-R31-R32;
(iv) R33-R31-R32-R32;
(v) R33-R31-R32-R32-R31; or
(vi) R33-R31-R32-R32-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y34)a-X34-(Z34)b, wherein X34, Y34, and Z34 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with a further embodiment, when the peptide includes the structure X35, the peptide may include the following structure:
Y35-X35, wherein X35 is as hereinabove described, and Y35 is:
(i) R33;
(ii) R32-R33;
(iii) R32-R32-R33;
(iv) R31-R31-R32-R32-R33;
(v) R32-R31-R32-R32-R33; or
(vi) R32-R32-R31-R32-R32-R33, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X35, the peptide may include the following structure:
X35-Z35, wherein X35 is as hereinabove described, and Z35 is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R31;
(v) R31-R32-R32-R31-R32; or
(vi) R31-R32-R32-R31-R32-R32, wherein R31 and R32 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y35)a-X35-(Z35)b, wherein X35, Y35, and Z35 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with a further embodiment, when the peptide includes the structure X36, the peptide may include the following structure:
Y36-X36, wherein X36 is as hereinabove described, and Y36 is:
(i) R31;
(ii) R33-R31;
(iii) R32-R33-R31;
(iv) R32-R32-R33-R31;
(v) R31-R32-R32-R33-R31; or
(vi) R32-R31-R32-R32-R33-R31, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X36, the peptide may include the following structure:
X36-Z36, wherein X36 is as hereinabove described, and Z36 is:
(i) R32;
(ii) R32-R32;
(iii) R32-R32-R31;
(iv) R32-R32-R31-R32;
(v) R32-R32-R31-R32-R32; or
(vi) R32-R32-R31-R31-R32-R33, wherein R31, R32, and R33 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y36)a-X36-(Z36)b, wherein X36, Y36, and Z36 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with one embodiment, when the peptide includes the structure X37, the peptide may include the structure Y37-X37, wherein X37 is as hereinabove described, and Y37 is:
(i) R32;
(ii) R31-R32;
(iii) R33-R31-R32 
(iv) R32-R33-R31-R32;
(v) R32-R32-R33-R31-R32; or
(v) R31-R32-R32-R33-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with a further embodiment, when the peptide includes the structure X37, the peptide may include the following structure:
X37-Z37, wherein X37 is as hereinabove described, and Z37 is:
(i) R32;
(ii) R32-R31;
(iii) R32-R31-R32;
(iv) R32-R31-R32-R32;
(v) R32-R31-R32-R32-R33; or
(vi) R32-R31-R32-R32-R33-R31, wherein R31, R32, and R33 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y37)a-X37-(Z37)b, wherein X37, Y37, and Z37 are as previously defined, a is 0 or 1, and b is 0 or 1.
In a preferred embodiment of the peptides according to formulae X31 to X37, n is 3, and most preferably the peptide is of one of the following structures as given in the accompanying sequence listing:
In (SEQ ID NO:67) and (SEQ ID NO:68), Xaa is p-aminophenylalanine.
In accordance with another embodiment, X is a peptide which includes the following basic structure X40:
R31-R32-R32-R33-R34-R32-R32-R31-R32-R32-R32-R34-R32,
wherein R31, R32, and R33 are as hereinabove described, and R34 is a basic hydrophilic or hydrophobic amino acid.
In accordance with one embodiment, the peptide may include the following structure:
Y40-X40, wherein X40 is as hereinabove described, and Y40 is:
(i) R32;
(ii) R32-R32;
(iii) R34-R32-R32 
(iv) R33-R34-R32-R32;
(v) R32-R33-R34-R32-R32;
(vi) R32-R32-R33-R34-R32-R32; or
(vii) R31-R32-R32-R33-R34-R32-R32, wherein R31, R32, R33, and R34 are as hereinabove described.
In accordance with another embodiment, X is a peptide which includes the following structure:
X40-Z40, wherein X40 is as hereinabove described and Z40 is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R33;
(v) R31-R32-R32-R33-R34;
(vi) R31-R32-R32-R33-R34-R32; or
(vii) R31-R32-R32-R33-R34-R32-R32, wherein R31, R32, R33, and R34 are as hereinabove described.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y40)a-X40-(Z40)b, wherein X40, Y40, and Z40 are as previously defined, a is 0 or 1, and b is 0 or 1.
In a preferred embodiment, the peptide has the following structural formula as given in the accompanying sequence listing:
(SEQ ID NO: 69)
In another preferred embodiment, the peptide has the following structural formula as given in the accompanying sequence listing:
(SEQ ID NO: 70)
In accordance with a further embodiment, the peptide has one of the following structural formulae as given in the accompanying sequence listing:
(SEQ ID NO: 71)
(SEQ ID NO: 72)
(SEQ ID NO: 73)
(SEQ ID NO: 74)
(SEQ ID NO: 75)
(SEQ ID NO: 76)
(SEQ ID NO: 77)
(SEQ ID NO: 78)
(SEQ ID NO: 79)
(SEQ ID NO: 80)
(SEQ ID NO: 81)
(SEQ ID NO: 82)
(SEQ ID NO: 83)
(SEQ ID NO: 84)
(SEQ ID NO: 85)
In accordance with another embodiment, X is a peptide which includes one of the following structural formulae:
(i)xe2x80x94(Lys Ile Ala Lys Lys Ile Ala)n-,
(ii)xe2x80x94(Lys Phe Ala Lys Lys Phe Ala)n-, and
(iii)xe2x80x94(Lys Phe Ala Lys Lys Ile Ala)n-, wherein n is from 1 to 5. Preferably, n is 3, and the peptide has one of the following structural formulae:
(Lys Ile Ala Lys Lys Ile Ala)3 (SEQ ID NO: 86)
(Lys Phe Ala Lys Lys Phe Ala)3 (SEQ ID NO: 87)
(Lys Phe Ala Lys Lys Ile Ala)3 (SEQ ID NO: 88)
In accordance with another embodiment, the X is a peptide which is selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
(SEQ ID NO: 89)
(SEQ ID NO: 90)
(SEQ ID NO: 91)
(SEQ ID NO: 92)
In accordance with yet another embodiment, X is a cecropin or sarcotoxin.
The term xe2x80x9ccecropinxe2x80x9d includes the basic structure as well as analogues and derivatives thereof. The cecropins and analogues and derivatives thereof are described in Ann. Rev. Microbiol., 1987, Vol. 41, pages 103-26, in particular page 108, and in Christensen, et al., PNAS, Vol. 85, pgs. 5072-76, which are hereby incorporated by reference.
The term xe2x80x9csarcotoxinxe2x80x9d includes the basic materials as well as analogues and derivatives thereof. The sarcotoxins and analogues and derivatives thereof are described in Molecular Entomology, pages 369-78, in particular page 375, Alan R. Liss, Inc. (1987), which is hereby incorporated by reference.
In accordance with another embodiment, X is melittin or an analogue or derivative thereof. Melittin is an amphipathic peptide consisting of 26 amino acid residues, and is isolated from honeybee (Apis mellifera) venom. Habermann, et al., Hoppe-Seyler""s Zeitschrift Physiol. Chem., Vol. 348, pgs. 37-50 (1987) (which document is entirely incorporated herein by reference). Melittin has the following structural formula as represented by the three-letter amino acid code:
In another embodiment, X is an amphiphilic peptide which is a hybrid of a cecropin peptide and a melittin peptide or an analogue thereof. Such hybrid peptides are described in U.S. Pat. No. 5,714,467 which is herein incorporated by reference. An example of such a peptide is given below.
I Lys-Ala-Lys-Leu-Phe-Ala-Lys-Ala-Gly-Ala-Gly-Ala-Val-Leu-Lys-Ala-Leu-Lys-Lys-Gly-Leu-Lys-Ala-Leu-Ile-Lys (SEQ ID NO: 156) (xe2x80x94OH or xe2x80x94NH2)
In another embodiment, X is an amphiphilic peptide which includes the following basic structure X50:
R41-R42-R42-R41-R42-R42-R41-R41-R42-R41-R41.
R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment, the peptide includes the basic structure Y50-X50, wherein X50 is as hereinabove described and Y50 is:
(I) R41;
(ii) R42-R41; or
(iii) R42-R42-R41, wherein R41 and R42 are as hereinabove described.
In one embodiment, R41 is leucine. In another embodiment, R42 is lysine. Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)
(SEQ ID NO: 97)
In accordance with another embodiment, X is an amphiphilic peptide which includes the following basic structure X52:
R42-R41-R42-R42-R41-R41-R42-R42-R41-R42-R42, wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment R41 is leucine. In another embodiment, R42 is lysine.
In one embodiment, the peptide includes the basic structure Y52-X52, wherein X52 is as hereinabove described, and Y52 is:
(I) R42;
(ii) R41-R42;
(iii) R41-R41-R42;
(iv) R42-R41-R41-R42; or
(v) R42-R42-R41-R41-R42, wherein R41 and R42 are as hereinabove described.
In one embodiment, the peptide may have the following structure:
(SEQ ID NO.: 98)
In another embodiment, the peptide includes the basic structure X52-Z52, wherein X52 is as hereinabove described, and Z52 is:
(I) R41;
(ii) R41-R41;
(iii) R41-R41-R42;
(iv) R41-R41-R42-R42; or
(v) R41-R41-R42-R42-R41, wherein R41 and R42 are as hereinabove described.
In one embodiment, the peptide may have the following structure:
(SEQ ID NO: 99)
In another embodiment, the peptide may include the structure:
(Y52)a-X52-(Z52)b, wherein X52, Y52, and Z52 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
In another embodiment, X is a biologically active amphiphilic peptide which includes the following basic structure X54:
R41-R42-R42-R41-R41-R42-R42-R41-R42-R42-R41-R41-R42-R42-R42-R43;
wherein R41 and R42 are as hereinabove described, and R43 is a neutral hydrophilic amino acid.
In one embodiment, the peptide may have the following structure:
(SEQ ID NO: 100)
In another embodiment, the peptide may have the following structure:
(SEQ ID NO: 101)
In another embodiment, X is a biologically active amphiphilic peptide which includes the following basic structure X56;
R41-R42-R42-R41-R41-R42-R42-R41 -R41-R42-R42-R44, wherein R/41 and R42 are as hereinabove described, and R44 is a neutral hydrophilic amino acid or proline.
In one embodiment, the peptide may include the structure:
X56-Z56wherein X56 is as hereinabove described, and Z56 is:
(I) -R42;
(ii) -R42-R42;
(iii) -R42-R42-R41;
(iv) -R42-R42-R41-R41;
(v) -R42-R42-R41-R41-R42;
(vi) -R42-R42-R41-R41-R42-R42; or
(vii) -R42-R42-R41-R41-R42-R42-R41, wherein R41 and R42 are as hereinabove described.
In a preferred embodiment, the peptide may have one of the following structures:
(SEQ ID NO: 102); or
(SEQ ID NO: 103).
In another embodiment, X is a biologically active amphiphilic peptide which includes the following basic structure X58;
R41-R41-R42-R42-R41-R42-R42-R41-R41-R42-R42-R41-R43,
wherein R41, R42, and R43 are as hereinabove described.
In one embodiment, the peptide includes the structure X58-Z58, wherein X58 is as hereinabove described, and Z58 is:
(I) -R41;
(ii) -R41-R45;
(iii) -R41-R45-R45;
(iv) -R41-R45-R45-R43;
(v) -R41-R45-R45-R43-R41;
(vi) -R41-R45-R45-R43-R41-R43;
(vii) -R41-R45-R45-R43-R41-R43-R43;
(viii) -R41-R45-R45-R43-R41-R43-R43-R45; or
(ix) -R41-R45-R45-R43-R41-R43-R43-R45-R43, wherein R41 and R43 are as hereinabove described, and R45 is proline.
In one embodiment, the peptide has the following structure:
(SEQ ID NO: 104).
In another embodiment, X is a biologically active amphiphilic peptide which includes the following basic structure X60:
R41-R41-R43-R42-R41-R41-R41-R41-R41-R41-R42-R41-R41-R42-R42-R41-R41-R42-R42-R41-, wherein R41, R42, and R43 are as hereinabove described. In one embodiment, the peptide may have the following structure:
(SEQ ID NO: 105).
In accordance with another embodiment, X is a peptide which includes the following basic structure X62:
-R41-R42-R42-R41-R42-R42-R41-,
wherein R41 and R42 are as hereinabove described.
In one embodiment, the peptide includes the following structure Y62-X62 where X62 is as hereinabove described, and Y62 is
(I) -R11;
(ii) -R42-R41;
(iii) -R42-R42-R41; or
(iv) -R41-R42-R42-R41, wherein R41 and R42 are as hereinabove described.
Representative examples of such peptides include the following, the sequences of which are given in the accompanying sequence listing:
(SEQ ID NO: 106)
(SEQ ID NO: 107)
(SEQ ID NO: 108)
(SEQ ID NO: 109)
(SEQ ID NO: 110)
(SEQ ID NO: 111)
(SEQ ID NO: 141)
(SEQ ID NO: 142)
(SEQ ID NO: 143)
(SEQ ID NO: 144)
(SEQ ID NO: 145)
(SEQ ID NO: 146)
In one embodiment, the peptide includes the structure X62-Z62, wherein X62 is as hereinabove described, and Z62 is:
(I) -R41;
(ii) -R41-R42;
(iii) -R41-R42-R42; or
(iv) -R41-R42-R42-R41, where R41 and R42 are as hereinabove described.
A representative example includes the following peptide having the structural formula given below and listed in the accompanying sequence listing:
(SEQ ID NO: 112)
In another embodiment, the peptide has the structure (Y62)a-X62-(Z62)b, wherein X62, Y62, and Z62 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
Representative examples of such peptides include the following, the structures of which are given in the accompanying sequence listing:
(SEQ ID NO: 113)
(SEQ ID NO: 114)
(SEQ ID NO: 115)
(SEQ ID NO: 116)
In another embodiment, X is a peptide having the following structural formula:
(SEQ ID NO: 117)
In another embodiment, X is a biologically active amphiphilic peptide including the following basic structure X64:
-R42-R42-R42-R41-R41-R42-R42-R41-,
wherein R41 and R42 are as hereinabove described.
In one embodiment, the peptide may include the structure Y64-X64, wherein X64 is as hereinabove described, and Y64 is:
(I) R41; or
(ii) R42-R41, wherein R41 and R42 are as hereinabove described.
In another embodiment, the peptide may include the structure X64-Z64, wherein X64 is as hereinabove described, and Z64 is:
(I) R42;
(ii) R42-R42; or
(iii) R42-R42-R41, wherein R41 and R42 are as hereinabove described.
In yet another embodiment, the peptide has the structure:
(Y64)a-X64-(Z64)b, wherein X64, Y64, and Z64 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
Representative examples of such peptides include the following:
(SEQ ID NO: 127)
(SEQ ID NO: 128)
(SEQ ID NO: 129)
In yet another embodiment, X is a biologically active amphiphilic peptide including the following basic structure X66:
R41-R42-R42-R41-R41-R41-R42-R41-R42-R42-R41, wherein R41 and R42 are hereinabove described and R46 is glutamic acid. A representative example of such a peptide is the following:
(SEQ ID NO: 130)
In yet another embodiment, X is a biologically active amphiphilic peptide including the following basic structure X68:
-R42-R42-R41-R41-R42-R46-R41-R42-R42-R41-, wherein R41, R42, and R46 are hereinabove described.
In one embodiment, the peptide includes the following basic structure Y68-X68, wherein X68 is as hereinabove described, and Y68 is R41 (defined above).
Representative examples of such peptides include the following:
(SEQ ID NO: 131)
(SEQ ID NO: 132).
In another embodiment, X is a biologically active amphiphilic peptide including the following basic structure X70:
-R41-R42-R42-R41-R41-R42-R42-R41-R42-R42-R41-R41-, wherein R41 and R42 are hereinabove described. A representative example of such a peptide has the following structure:
(SEQ ID NO: 133).
In another embodiment, X is a biologically active amphiphilic peptide including the following basic structure X72:
-R42-R12-R41-R41-R42-R47-R41-R42-R42-R41-, wherein R41 and R42 are hereinabove described, and R47 is aspartic acid. A representative example of such a peptide has the following structure:
(SEQ ID NO: 134).
In yet another embodiment, X is a biologically active amphiphilic peptide having the following structure:
(SEQ ID NO: 135).
In yet another embodiment, X is a biologically active amphiphilic peptide including the following structure X74:
R42-R41-R42-R41-R41-R42-R42-R41-R46-R42-R41, wherein R41, R42, and R46 are hereinabove described. A representative example of such a peptide has the following structure:
(SEQ ID NO: 136).
In another embodiment, X is a biologically active amphiphilic peptide including the following structure X76:
-R41-R42-R42-R41-R41-R42-, wherein R41 and R42 are hereinabove described.
In another embodiment, the peptide includes the structure Y76-X76-, wherein X76 is as hereinabove described, and Y76 is:
(I) -R42;
(ii) -R42-R42;
(iii) -R41-R42-R42;
(iv) -R41-R41-R42-R42;
(v) -R42-R41-R41-R42-R42; or
(vi) -R42-R42-R41-R41-R42-R42, wherein R41 and R42 are as hereinabove described.
In another embodiment, the peptide includes the structure X76-Z76, wherein X76 is as hereinabove described, and Z76 is:
(I) R48;
(ii) R49-R41; or
(iii) R48-R41-R42, wherein R41 and R42 are as hereinabove described, and R48 is a basic hydrophilic, neutral hydrophilic, or hydrophobic amino acid.
In yet another embodiment, the peptide has the following structural formula:
(Y76)a-X76-(Z76)b, wherein X76, Y76, and Z76 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
Representative examples of such peptides include the following:
(SEQ ID NO: 137)
(SEQ ID NO: 138)
(SEQ ID NO: 139).
In yet another embodiment, X is a biologically active amphiphilic peptide including the following structural formula X78:
-R41-R42-R41-R41-R42-R42-R41-R42-R42-R41, wherein R41 and R42 are as hereinabove described. A representative example of such a peptide has the following structure:
(SEQ ID NO: 140).
In another embodiment, X has the following structure:
(SEQ ID NO: 149).
In another embodiment, X is a biologically active amphiphilic peptide including the following structural formula X80:
-R41-R42-R42-R41-R41-R42-R46-R41-R41-R42-R41-, wherein R41, R42, and R46 are as hereinabove described. A representative example of such a peptide has the following structure:
(SEQ ID NO: 151).
In accordance with yet another embodiment, X is an ion channel-forming peptide or protein.
Ion channel-forming proteins or peptides which may be employed include defensins, also known as human neutrophil anti-microbial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), and a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein. Defensins are described in Selsted, et al., J. Clin. Invest., Vol. 76, pgs. 1436-1439 (1985). MBP proteins are described in Wasmoen, et al., J. Biol. Chem., Vol. 263, pgs. 12559-12563 (1988). BPI proteins are described in Ooi, et al., J. Biol. Chem., Vol. 262, pgs. 14891-14894 (1987). Perforin is described in Henkart, et al., J. Exp. Med., 160: 75 (1984), and in Podack, et al., J. Exp. Med., 160:695 (1984). The above articles each are entirely incorporated herein by reference.
The term xe2x80x9cion channel-forming proteinsxe2x80x9d includes the basic structures of the ion channel-forming proteins as well as analogues and derivatives.
In accordance with yet another embodiment, each of the amino acid residues of the peptides or proteins may be a D-amino acid or glycine. Although the scope of this particular embodiment is not to be limited to any theoretical reasoning, it is believed that the above-mentioned peptides or proteins, when consisting entirely of D-amino acid or glycine residues, may have increased resistance to proteolytic enzymes while retaining their activity. Such peptides thus may be administered orally. Also, in accordance with another embodiment, all of the amino acid residues may be D-amino acid or glycine residues, or L-amino acid or glycine residues.
It is also to be understood that the peptides or proteins may be administered in combination with one another.
In accordance with another embodiment, the N-terminal substituted peptides or proteins of the present invention may be employed in combination with an ion having pharmacological properties for the purposes hereinabove described.
An ion having pharmacological properties is one which, when introduced into a target cell, virus, or virally-infected cell, inhibits and/or prevents and/or destroys the growth of the target cell, virus, or virally-infected cell.
Such an ion having pharmacological properties is one which, in the absence of an ion channel forming peptide, is unable to cross a natural or synthetic lipid membrane, in particular a cell or virus membrane, in sufficient amounts to affect a cell or virus adversely.
The peptide or protein and ion having pharmacological properties may be administered as a single composition or in separate compositions, and the single or separate compositions may include additional materials, actives, and/or inactives, in addition to the peptide or protein and ion having pharmacological properties. As representative examples of ions having pharmacological properties which may be employed, there may be mentioned fluoride, peroxide, bicarbonate, silver, zinc, mercury, arsenic, copper, platinum, antimony, gold, thallium, nickel, selenium, bismuth, and cadmium ions.
The peptide or protein and the ion having pharmacological properties, whether administered or prepared in a single composition or in separate compositions, are employed in amounts effective to inhibit and/or prevent and/or destroy the growth of the target cell, virus, or virally-infected cell. In effect, the ion potentiates the action of the peptide, i.e., the amount of ion is effective to reduce the maximum effective concentration of the peptide or protein for inhibiting growth of a target cell, virus, or virally-infected cell.
The ion having pharmacological properties, when used topically, is generally employed in a concentration of from 0.05% to 2.0%. When used systemically, the ion is generally employed in an amount of from 1 to 10 mg. per kg. of host weight. Peptide or protein dosages may be within the ranges hereinabove described.
It is also to be understood that the peptide or protein and ion having pharmacological properties, may be delivered or administered in different forms. For example, the ion may be administered orally, while the peptide may be administered by IV or IP.
As representative examples of administering the peptide or protein and ion for topical or local administration, the peptide could be administered in an amount of up to about 1% weight to weight and the ion delivered in an amount of about 50 mM (about 0.1%). Alternatively, the ion, in the form of a salt such as sodium fluoride, could be administered orally in conjunction with systemic administration of the peptide or protein. For example, the peptide or protein may be administered IV or IP to achieve a serum dose of 100 micrograms per milliliter (10 milligrams per kilogram) in conjunction with an oral dose of ion, in particular, sodium fluoride, of 10 meq per kilogram.
In accordance with another embodiment, the peptides or proteins of the present invention may be administered to a host in combination with an antibiotic selected from the class consisting of bacitracins, gramacidin, polymyxin, vancomycin, teichoplanin, aminoglycosides, hydrophobic antibiotics, penicillin, monobactams, or derivatives or analogues thereof.
The bacitracins, gramacidin, polymyxin, vancomycin, teichoplanin, and derivatives and analogues thereof, are a group of polypeptide antibiotics. A preferred bacitracin is bacitracin A.
Aminoglycoside antibiotics include tobramycin, kanamycin, amikacin, the gentamicine (e.g., gentamicin C1, gentamicin C2, gentamicin C1a), netilmicin, and derivatives and analogues thereof. The preferred aminoglycosides are tobramycin and the gentamicins. The aminoglycosides, and the bacitracins hereinabove described, tend to be hydrophilic and water-soluble.
Penicillins which may be employed include, but are not limited to, benzyl penicillin, ampicillin, methicillin (dimethoxyphenyl penicillin), ticaricillin, penicillin V (phenoxymethyl penicillin), oxacillin, cloxacillin, dicloxacillin, flucloxacillin, amoxicillin, and amidinocillin. Preferred penicillins which may be employed are benzyl penicillin and ampicillin. A preferred monobactam which may be employed is aztreonam.
As representative examples of hydrophobic antibiotics which may be used in the present invention, there may be mentioned macrolides, such as erythromycin, roxythromycin, clarithromycin, etc.; 9-N-alkyl derivatives of erythromycin; midecamycin acetate; azithromycin; flurithromycin; rifabutin; rokitamycin; a 6-0-methyl erythromycin A known as TE-031 (Taisho); rifapentine; benzypiperazinyl rifamycins such as CGP-7040, CGP-5909, CGP-279353 (Ciba-Geigy); an erythromycin A derivative with a cyclic carbamate fused to the C11/C12 position of a macrolide ring known as A-62514 (Abbott); AC-7230 (Toyo Jozo); benzoxazinorifamycin; difficidin; dirithromycin; a 3-N-piperdinomethylzaino methyl rifamycin SV known as FCE-22250 (Farmitalia); M-119-a (Kirin Brewery); a 6-0-methyl-1-4xe2x80x3-O-carbamoyl erythromycin known as A-63075 (Abbott); 3-formylrifamycin SV-hydrazones with diazabicycloalkyl side chains, such as CGP-27557 and CGP-2986 (Ciba-Geigy); and 16-membered macrolides having a 3-0-alpha-L-cladinosyl moiety, such as 3-0-alpha-L-cladinosyldeepoxy rosaramicin; tylosins and acyl demycinosyl tylosins.
In addition to the macrolides hereinabove described, rifamycin, carbenicilllin, and nafcillin may be employed as well.
Other antibiotics which may be used (whether or not hydrophobic) are antibiotics which are 50-S ribosome inhibitors such as lincomycin; clindamycin; and chloramphenicol; etc.; and antibiotics which have a large lipid like lactone ring, such as mystatin; pimaricin, etc.
The peptide or protein and antibiotic may be administered by direct administration to a target cell by systemic or topical administration to a host which includes the target cell, in order to prevent, destroy, or inhibit the growth of a target cell. Target cells whose growth may be prevented, inhibited, or destroyed by the administration of the peptides and antibiotic include Gram-positive and Gram-negative bacteria, as well as fungal cells.
The antibiotic, such as those hereinabove described, or derivatives or analogues thereof, when used topically, is generally employed in a concentration of about 0.1% to about 10% (by weight). When used systemically, the antibiotic or derivative or analogue thereof is generally employed in an amount of from 1.25 mg. to about 45 mg. per kg. of host weight per day. Peptide or protein dosages may be those as hereinabove described.
As representative examples of administering the peptide or protein and antibiotic for topical or local administration, the peptide or protein could be administered in an amount of from 0.1% to about 10% weight to weight, and the antibiotic is delivered in an amount of from about 0.1% to about 10% weight to weight.
In accordance with another embodiment, the peptides or proteins of the present invention may be administered in combination with an anti-parasitic agent or an anti-fungal agent.
Anti-parasitic agents which may be employed include, but are not limited to, anti-protozoan agents. Examples of specific anti-parasitic agents which may be employed include, but are not limited to, pentamidine isethionate, and propamidine isethionate (Brolene).
Anti-fungal agents which may be employed include, but are not limited to, ketoconazole. It is also to be understood that certain anti-parasitic agents may also have anti-fungal activity, and that anti-fungal agents may have anti-parasitic activity.
In accordance with another embodiment, the peptides or proteins of the present invention may be administered in combination with an antibiotic which inhibits DNA gyrase, which is an enzyme involved in the formation of bonds between individual coiling strands of replicating bacterial DNA. Thus, DNA gyrase is necessary for the normal replication of bacterial DNA, and, therefore, antibiotics which inhibit DNA gyrase inhibit the normal replication of bacterial DNA.
Examples of antibiotics which inhibit DNA gyrase include nalidixic acid, oxolinic acid, cinoxacin, and quinolone antibiotics which include ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxacin, fleroxacin, tosulfoxacin, temafloxacin, and rufloxacin.